Engineered Protein-G variants for plug-and-play applications.

Area of Science:

• Biotechnology
• Molecular Biology
• Immunology

Background:

• Antibody engineering is crucial for developing advanced diagnostics and therapeutics.
• Current methods for creating multifunctional antibody assemblies can be time-consuming and complex.
• There is a need for versatile, modular systems to streamline antibody-based reagent production.

Purpose of the Study:

• To develop a modular, plug-and-play platform for assembling multifunctional antibody-based reagents.
• To engineer high-specificity antibody fragments (Fabs) and protein G (PG) variants for orthogonal pairing.
• To demonstrate the platform's utility in creating diverse antibody formats, including IgG-like assemblies, bispecific antibodies, and Bispecific T-cell Engagers (BiTEs).

Main Methods:

• Utilized phage display to engineer specific Fab-PG pairings.
• Developed prefabricated antibody modules and PG variants fused to SNAP-tags for multiplexed detection.
• Constructed IgG-like assemblies and bispecific antibodies using PG-Fc dimer core units and the "knobs into holes" strategy.
• Incorporated commercial antibodies as building blocks.
• Performed crystal structure analysis to understand specificity-determining interactions.

Main Results:

• Successfully created a portfolio of antibody modules with high pair-wise specificity.
• Demonstrated rapid assembly of customized IgG-like antibodies and bispecific analogs in minutes.
• Showcased the application of bispecific modalities in fabricating potent Bispecific T-cell Engagers (BiTEs) with demonstrated cancer cell-killing efficacy.
• Confirmed the system's adaptability to include commercial antibodies, expanding target accessibility.
• Structural analysis revealed key interactions responsible for scaffold specificity.

Conclusions:

• The developed plug-and-play antibody module platform offers a user-friendly and versatile approach for creating complex antibody assemblies.
• This system significantly accelerates the production of customized antibody reagents, reducing timelines from weeks to minutes.
• The platform holds substantial promise for advancing cell biology research, diagnostics, and therapeutic development through enhanced antibody functionality.